Project Summary Mantle cell lymphoma (MCL) is a B cell non-Hodgkin's lymphoma with an overall poor prognosis, and is currently incurable due to the eventual development of drug resistance. Unrestrained proliferation driven by cyclin D1 overexpression often underlies disease progression. The first phase I, single-agent clinical trial targeting the cyclin-dependent kinases CDK4/CDK6 with palbociclib treatment in recurrent MCL resulted in a durable clinical response, with tumor regression in some MCL patients. In ongoing clinical trials, palbociclib, in combination with other drugs targeting MCL survival is showing remarkable therapeutic efficacy, with many patients achieving a complete response at reduced doses. However, the mechanism by which CDK4/6 inhibition improves the efficacy of other targeted agents remains to be defined. Understanding this mechanism is critical to improving the response to these agents and overcoming resistance. In collaborative clinical and mechanistic studies with Project 1, we discovered that inhibition of CDK4/6 by palbociclib leads to prolonged early G1 arrest (pG1), which sensitizes cancer cells to cytotoxic killing by BTK or PI3K inhibition. This requires the action of FOXO1 transcription factor, which is activated and localized to the nucleus in pG1. Tumor suppressor FOXO1 is a central component of the PI3K signaling cascade engaged by the B cell receptor, and is essential for B cell homeostasis. We showed that FOXO1 expression and nuclear localization are necessary for cytotoxic killing by palbociclib in combination with a BTK or PI3K inhibitor. We found that pG1 induced repressive chromatin remodeling by the polycomb repressive complex 2 (PRC2), and that its perturbation induces synergistic killing of MCL cells in pG1. We hypothesize that pG1 induction by CDK4/6 inhibition in MCL cells causes specific epigenetic alterations that modify FOXO1 access to its target genes, altering FOXO1- mediated cytotoxic gene expression. We predict that timely perturbation of these epigenetic events should increase FOXO1 target gene expression, thus enhancing clinical outcomes of palbociclib and combined cytotoxic agent-induced killing. Hence our proposal will define the mechanisms underlying cytotoxic killing of MCL by targeting the cell cycle, and its coordinately regulated epigenetic and transcriptional machinery with specific aims (1) to elucidate the role of PRC2 in chromatin remodeling in pG1, and (2) to define the role of FOXO1 in CDK4 inhibitor sensitization to BTK or PI3K inhibition. Identification of downstream gene targets of FOXO1, and the cellular processes that they support, especially those that mediate cytotoxicity, will advance the rational design of mechanism-based, effective, and durable cancer therapies.